HUMANIZED CD37 AND Bl-SPECIFIC CD19-HUMANIZED CD37 CAR-T CELLS

ABSTRACT

The present invention is directed to humanized CD37-CAR comprising humanized CD37 scFv of the present invention. The present invention is also directed to a bispecific CD19-humanized CD37 CAR, comprising: (i) CD19 VL, (ii) Humanized CD37 ScFv, (iii) CD19 VH, (iv) a transmembrane domain, (v) at least one co-stimulatory domains, and (vi) an activating domain. The CARs of the present invention are useful in the field of adoptive immunity gene therapy for hematological cancers.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with the specification as an ASCII formatted text file via EFS-Web with a file name of SequenceListing.txt with a creation date of Jul. 21, 2021, and a size of 49.5 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to humanized CD37 CAR and bi-specific CD19-humanized CD37-CAR and CAR-T cells, which are useful in the field of adoptive immunity gene therapy for hematological cancers.

BACKGROUND OF THE INVENTION

Immunotherapy is emerging as a highly promising approach for the treatment of cancer. T cells or T lymphocytes, the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells. Genetically modifying T cells with CAR (Chimeric antigen receptor) constructs is the most common approach to design tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach [1, 2]. The advantage of CAR-T technology compared with chemotherapy or antibody is that reprogrammed engineered T cells can proliferate and persist in the patient (“a living drug”) [1], [3].

CARs usually consist of a monoclonal antibody-derived single-chain variable fragment (scFv) at the N-terminal part, hinge, transmembrane domain and a number of intracellular co-activation domains: (i) CD28, (ii) CD137 (4-1BB), CD27 or other co-stimulatory domains, in tandem with a activation CD3-zeta domain. (FIG. 1 ) [1; 2]. The evolution of CARs went from first generation (with no co-stimulation domains) to second generation (with one co-stimulation domain) to third generation CAR (with two co-stimulation domains) and fourth generation (with several co-stimulation domains). Generating CARs with two costimulatory domains (the so-called 3^(rd) generation CAR) have led to increased cytolytic CAR-T cell activity, improved persistence of CAR-T cells leading to its augmented antitumor activity.

FIG. 1 show the structures of CAR. The left panel shows the structure of the first generation (no co-stimulation domains). The middle panel shows the structure of the second generation (one co-stimulation domain CD28 or 4-BB). The right panel shows a third generation of CAR (two or several co-stimulation domains). The Figure is from Golubovskaya, Wu, Cancers, 2016 [3].

CD37 is a 40-52 kDa heavily glycosylated member of the transmembrane 4 superfamily (TM4SF) of tetraspanin proteins. CD37 plays a role in integrin, AKT, PI3-Kinase-dependent survival, and apoptotic signaling, motility, immune response signaling via activation of dendritic cell migration.

CD37 is highly expressed in many hematological cancers, such as non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and in some peripheral and cutaneous T cell lymphomas, and absent or weakly expressed in multiple myeloma and Hodgkin's lymphoma.

CD19 is a known antigen overexpressed in B-cell malignant cancers such as leukemia and lymphoma. CD37 is a member of tetraspanin (or transmembrane 4 superfamily (TM4SF) protein) family proteins, which have four potential membrane-spanning regions, CD37 is also overexpressed in leukemia and lymphoma patients [4].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . The structures of CAR.

FIGS. 2A and 2B show the structures of humanized CD37-CAR construct and bi-specific CD19-humanized CD37 CAR construct. FIG. 2A. Humanized CD37-CAR construct. Signal peptide (CD8 alpha leader sequence); humanized CD37 ScFv (VH-linker-VL); CD8 hinge; CD28 transmembrane domain; 41BB costimulatory domain; CD3 activation domain. FIG. 2B. Bispecific CD19-humanized CD37 construct. The second-generation lentiviral CAR under MNDU3 promoter construct are used with 4-1BB co-stimulatory domain. GM-CSFR alpha signaling peptide, CD19 VL; Linker; Humanized CD37ScFv; Linker; CD19 VH, CD8 hinge; CD8 transmembrane domain, 41BB costimulatory and CD3 activation domains. Abbreviations: VL, light chain; L-linker; ScFv-single chain variable fragment; VH, heavy chain; H, hinge; TM, transmembrane domain; hCD37, humanized CD37 ScFv; 41BB co-stimulatory and CD3 activation domains are illustrated for CD19-hCD37 CAR.

FIGS. 3A-3C. The humanized CD37-CAR-T cells (PMC930) specifically target CD37-positive cells. FIG. 3A: Humanized CD37-CAR-T cells killed CHO-CD37-positive cells and did not kill CHO cells. Labeling of curves goes from top to bottom; TC-target cells; T cells, Mock CAR-T cells, and hCD37 CAR-T cells. FIG. 3B: Quantification of cytotoxicity shows significantly higher killing by CD37CAR-T cells in CHO-CD37 cells than Mock and T cells. *, hCD37 CAR-T cells with CHO-CD37 cells versus T and Mock CAR-T cells, p<0.0001, One-Way ANOVA followed by Dunnett's Multiple Comparison Test. FIG. 3C: hCD37-CAR-T cells secrete significantly higher IFN-gamma with CHO-CD37 cells than with CHO cells. *, p<0.05, IFN-gamma of humanized Hum CD37 CAR-T cells with CHO-CD37 cells versus same CAR-T cells with CHO cells by Student's t-test.

FIGS. 4A-4E. Bi-specific hCD37-CD19-CAR-T cells specifically target CD37-positive and CD19-positive cells. FIG. 4A: RTCA activity of hCD37-CD19-CAR-T cells with CHO-CD37 cells (left) and CHO cells (right). Cytotoxicity of bispecific CD37-CD19 CAR-T cells against CHO-CD37 cells was significantly higher than that of humanized CD37 CAR-T cells, *, p<0.0001, hCD37 and hCD37-CD19-CAR-T cells with CHO-CD37 cells vs T and Mock CAR-T cells, One-Way ANOVA followed by Sidak's multiple comparison test p=0.0006. FIG. 4B: RTCA activity of hCD37-CD19-CAR-T cells with Hela-CD19 cells (left) and Hela cells (right). Quantification of RTCA at the end time point is shown under the RTCA plots. * p<0.0001, * hCD37-CD19 CAR-T cells and CD19 CAR-T cells with Hela-CD19 cells vs T cells, Mock CAR-T cells, CD37 CAR-T cells by One-Way ANOVA followed by Sidak's multiple comparison as in B. FIG. 4C: IFNgamma secretion by hCD37-CD19-CAR-T cells was significantly higher with CHO-CD37 cells than with CHO cells. *p<0.0001, CD37, hCD37-CD19 CAR-T cells vs other groups with CHO-CD37 cells by One-way ANOVA followed by Tukey's test. FIG. 4D: IFN-gamma secretion by CD37-CAR-T cells was significantly higher with Hela-CD19 cells than with Hela cells, *p<0.05, hCD37-CD19 and CD19 CAR-T cells with Hela-CD19 cells vs other groups with Raji cells, Student's t-test. FIG. 4E: IFN gamma secretion by hCD37-CD19-CAR-T cells against Raji cells was significantly higher than with CD37-negative multiple myeloma MM1S cells, p<0.001, * hCD37, hCD37-CD19 and CD19 CAR-T cells with Raji cells vs Mock CAR-T cell groups with Raji cells by Tukey's test.

FIG. 5 . RTCA assay comparing PMC985, PMC930 and PMC910 and PMC1046 using Hela-CD19, Hela cell lines. Humanized CD37 CAR: PMC985 and PMC910; bispecific CD19-CD37 CAR PMC930 and PMC1046.

FIG. 6 . Humanized CD37 CAR-T cells significantly block Raji xenograft tumor growth in vivo. Humanized CD37 CAR-T cells significantly prolong mouse survival in Raji xenograft mouse model. p<0.0001, Kaplan-Myer survival vs negative control group.

FIGS. 7A-7C. hCD37-CD19-CAR-T cells significantly block Raji xenograft tumor growth in vivo. FIG. 7A: In vivo imaging of Raji tumors in mice on days 7, 14, and 21 following Raji-Luc+ cells injection with the vehicle, mock-CAR-T cells, or bispecific CD37-CD19 CAR-T cell-treated groups (n=5 each). FIG. 7B: Tumor luminescence flux from In Vivo Imaging System, IVIS imaging. Vehicle-treated mice had died by day 14. * p<0.05, hCD37-CD19 CAR-T cells (bottom curve) vs. Mock CAR-T cells, Student's t-test. FIG. 7C: hCD37-CD19-CAR-T cells significantly prolong mouse survival in the Raji xenograft model. Kaplan-Myer curve is shown, p<0.05, log-rank test hCD37-CD19 CAR-T cell-treated (far-right) vs. Mock CAR-T cell-treated group (middle).

BRIEF DESCRIPTION OF THE INVENTION Definitions

As used herein, a “chimeric antigen receptor (CAR)” is a receptor protein that has been engineered to give T cells the new ability to target a specific protein. The receptor is chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. CAR is a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. The “chimeric antigen receptor (CAR)” is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor (CIR).” The “extracellular domain capable of binding to an antigen” means any oligopeptide or polypeptide that can bind to a certain antigen. The “intracellular domain” means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

As used herein, a “domain” means one region in a polypeptide which is folded into a particular structure independently of other regions.

As used herein, “humanized antibodies” are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

As used herein, a “single chain variable fragment (scFv)” means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for engineering an scFv are known to a person skilled in the art.

As used herein, a “tumor antigen” means a biological molecule having antigenecity, expression of which causes cancer.

The present invention is directed to a humanized anti-CD37 antibody or an antigen-binding fragment thereof. The present invention is also directed to humanized CD37-CAR T cells that are effective in killing target cells.

The inventors have obtained a mouse monoclonal antibody specifically targeting human CD37 (WO2020/146267) and humanize it to make humanized anti-CD37 antibodies. The inventors then prepared humanized CD37-CAR T cells with the humanized anti-CD37 antibodies and tested them for effectiveness in killing target cells. The inventors tested CAR T cells prepared from 11 humanized anti-CD37 antibodies derived from the above-mentioned mouse monoclonal antibody, and selected two antibodies PMC762 and PMC910, which were the only two humanized antibodies that their CAR T cells were effective in killing target cells.

The advantages of humanized CD37 antibody versus mouse CD37 antibody include that humanized CD37 antibody does not cause immune response against mouse CD37 sequence, and humanized CD37 antibody is better tolerated inside humanized hCD37-CAR-T cells.

The present invention is directed to a humanized anti-human CD37 antibody or an antigen-binding fragment thereof (e.g., Fab, (Fab)₂, scFv), comprising (i) VH having the amino acid of SEQ ID NO: 3 and VL having the amino acid of SEQ ID NO: 4, or (ii) VH having the amino acid of SEQ ID NO: 8 and VL having the amino acid of SEQ ID NO: 9. In one embodiment, the antibody or an antigen-binding fragment thereof is a monoclonal antibody. In another embodiment, the antibody or an antigen-binding fragment thereof is a single-chain variable fragment (scFv). The scFv can be VH-linker-VL, or VL-linker-VH.

The present invention is also directed to a chimeric antigen receptor fusion protein CD37-CAR comprising from N-terminus to C-terminus: (i) humanized CD37 ScFv; (ii) a hinge; (iii) a transmembrane (TM) domain; (iv) at least one costimulatory domain; (v) one activation domain.

In another aspect, the present invention is directed to a bispecific CAR. Both CD37 and CD19 proteins play a role in survival signaling. Based on high percent of expression in B-cell lymphoma, both targets can be used for CAR-T cell therapy. Since CD19 expression can be down-regulated or lost in lymphoma patients due to alternatively spliced transcript or other mechanisms leading to patient relapse (2), bi-specific CD19-humanized CD37 can be used in CAR-T cell therapy.

The inventors obtained mouse monoclonal antibodies against human CD19 (FMC63[2]). The inventors then produced bi-specific CD19-humanized CD37 scFv-CAR-T cells to target cancer cells overexpressing CD19 and CD37 tumor antigen. The CD19-hCD37-CAR-T cells of the present invention have high and specific cytotoxic activity against CHO-CD19, CHO-CD37 cells, Hela-CD19, and Hela-CD37 cells.

The advantages of bi-specific CD19/hCD37 CARs include that the bi-specific CAR-T cells target both CD19 and CD37 antigens overexpressed in lymphoma, and therefore they are more effective. If one antigen (CD19 or CD37) is lost or down-regulated, the bi-specific CAR-T cells can still reach the other antigen.

The present invention is directed to a bispecific chimeric antigen receptor (CAR) fusion protein comprising from N-terminus to C-terminus: (i) CD19 VL, (ii) CD37 ScFv, (iii) CD19 VH; (iv) hinge; (v) a transmembrane domain, (vi) at least one co-stimulatory domains, and (vii) an activating domain.

In one embodiment, the humanized CD37-CAR structure is shown in FIG. 2A, and the bispecific CD19-hCD37-CAR is shown in FIG. 2B.

In FIG. 2A, the humanized CD37 scFc in CAR is shown as VH-linker-VL. Alternatively, the humanized CD37 scFc can be VL-linker-VH.

In FIG. 2B, the bispecific CD19-CD37 in CAR is shown as CD19VL-linker-CD37 ScFv-linker-CD19VH. Alternatively, the bispecific CD19-CD37 in CAR can be CD19VH-linker-CD37 ScFv-linker-CD19VL, or CD19ScFv-linker-CD37 ScFv, or CD37 ScFv-linker-CD19ScFv. The VH and VL arrangement in each ScFv can be VH-linker-VL or VL-linker-VH.

The linkers can be the same sequences or different sequences.

In one embodiment, the co-stimulatory domain if CAR is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP10. A preferred the co-stimulatory domain is CD28 or 4-1BB.

A preferred activating domain is CD3 zeta (CD3 Z or CD3ζ).

The transmembrane domain may be derived from a natural polypeptide, or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T cell receptor α or β chain, a CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or a polypeptide linker, for example, a linker having a length of 2 to 10 amino acids can be arranged between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence having a glycine-serine continuous sequence can be used.

The present invention provides a nucleic acid encoding the humanized CD37-CAR and the bispecific humanized CD37-CD19 CAR. The nucleic acid encoding the CAR can be prepared from an amino acid sequence of the specified CAR by a conventional method. A base sequence encoding an amino acid sequence can be obtained from the NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR).

A nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used. A virus vector lacking the replicating ability so as not to self-replicate in an infected cell is preferably used.

For example, when a retrovirus vector is used, a suitable packaging cell based on a LTR sequence and a packaging signal sequence possessed by the vector can be selected for preparing a retrovirus particle using the packaging cell. Examples of the packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. A retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.

A CAR-T cell binds to a specific antigen via the CAR, thereby a signal is transmitted into the cell, and as a result, the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of a host cell and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like as an index. For example, release of a cytotoxic cytokine (IFN-gamma, a tumor necrosis factor, lymphotoxin, etc.) from the activated cell causes destruction of a target cell expressing an antigen. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.

Humanized CD37-CAR-T cells can be used as allogenic CAR-T cells.

The cell expressing the CAR can be used as a therapeutic agent for a disease. The therapeutic agent comprises the cell expressing the CAR as an active ingredient, and it may further comprise a suitable excipient.

The inventors have generated hCD37-CAR-T cells and bi-specific CD19-hCD37-ScFv-41BB-CD3-CAR-T against hematological cancer cells overexpressing CD37 lymphoma. CD19-hCD37-CAR-T cells express higher specific cytotoxic activity against CD19-positive and CD37-positive target cancer cells than against non-transduced T cells and Mock-CAR-T cells.

The present humanized CD37 and bi-specific CD19-humanized CD37-CAR-T cells target CHO-CD19 and CHO-CD37 target cells but not CHO cells.

CD19-hCD37-CAR-T cells using the present CD19 and CD37 antibodies can be effectively used to target CD19 and CD37 antigens in CD19- and CD37-positive lymphoma.

CD19-hCD37-CAR-T can be used in combination with different therapies: checkpoint inhibitors; targeted therapies, small molecule inhibitors, and antibodies.

CD19-hCD37-CAR-T cells can be used clinically for targeting CD19- and CD37-positive cells.

Modifications of co-stimulating domains: CD28, 4-1BB and others can be used to increase its efficacy. Tag-conjugated CD19 ScFv or humanized CD37 ScFv can be used for CAR generation.

Third generation CAR-T or other co-activation signaling domains can be used for the same CD19-scFv inside CAR.

Humanized CD19 and humanized CD37 can be used for generation of CD19-CD37-CAR-T cells.

Combination of CD19-hCD37 ScFv-CAR with other CAR targeting other tumor antigens or tumor microenvironment (VEGFR-1-3), PDL-1, CD80 can be used to enhance activity of monotherapy CD19-CD37-CAR.

The present CD19-hCD37-CAR can be used to generate other types of cells such as CAR-natural killer (NK) cells, CD19-hCD37-CAR-macrophages, allogenic CAR-T cells, gene-edited T cells, and other CD19-hCD37-CAR hematopoietic cells, which can target CD19 and CD37-positive cancers. The present invention provides T cells, or NK cells, or macrophages, or hematopoietic cells, or other methods to down-regulate different markers of GVHD (graft versus host disease) and to express the CD37-CAR.

The present invention demonstrates the efficacy of CD37-CAR-T cells and bispecific hCD37-CD19 CAR-T cells in vitro and in vivo. CD37 has been identified as a possible target for NHL immunotherapy. The CD37-CAR-T cell therapy is especially important during lymphoma relapse when CD19 antigen is lost in lymphoma by either alternative splicing or other mechanisms, such as mutations. Thus, CD37-CAR-T cells can improve the outcome of CD19-negative relapsed lymphoma patients. Bi-specific CD37-CD19 CAR-T cells increase the efficacy of CD19-CAR-T cells and are important in case of CD37 antigen loss due to missense mutations or other mechanisms.

The present application shows that humanized CD37-CD19 CAR-T cells effectively blocked lymphoma growth in vivo that can be advantageous in case of downregulation of either CD19 or CD37 pathways or for more efficient targeting of both antigens.

Since lymphoma tumors are heterogeneous and surrounded by a microenvironment that can block immune response functions, the combination therapy of CAR-T cells with checkpoint inhibitors, checkpoint blocking antibodies with agonist antibodies inducing an immune response, or with small molecules can overcome these barriers.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLES Materials and Methods Example 1. Cell Lines, Antibodies, Recombinant Proteins

Raji, RPMI8226, H929, MM1S, K562, CHO, MCF-7, MDA-231, and Lovo cell lines were purchased from the ATCC (Manassas, Va., USA) and cultured either in DMEM (GE Healthcare, Chicago, Ill., USA) or in RPMI-1640 medium (ThermoFisher, Waltham, Mass., USA) containing 10% FBS (AmCell, Mountain View, Calif., USA). CHO-CD37 cells were purchased from BPS Bioscience (San Diego, Calif., USA) and cultured in Ham's F12K medium containing 10% Fetal Bovine Serum, FBS and 1 mg/mL geneticin (ThermoFisher). Hela-CD37 were generated by transducing Hela cells with CD37 lentivirus. Human peripheral blood mononuclear cells (PBMC) from whole blood obtained in the Stanford Hospital Blood Center, Stanford, according to IRB-approved protocol (#13942), were isolated by density sedimentation over Ficoll-Paque (GE Healthcare, San Ramon, Calif., USA).

Recombinant proteins CD37, CD318, GATA3, CD89, CD43, SP10, MSH2, SERPINA1 were obtained from Promab (Richmond, Calif., USA). For ELISA with CD37 and other proteins, HRP labeled anti-Mouse IgG was used from Sigma-Aldrich (St Louis, Mo., USA) (Cat #: A0168). Human serum and goat anti-mouse (Fab)₂ or anti-human (Fab)₂, CD3 antibodies for FACS were from Jackson Immunoresearch (West Grove, Pa., USA).

Example 2. Humanization of Mouse Anti-CD37 VH and VL

Humanization of mouse CD37 VH and VL (see WO2020/146267) was performed as described before [5, 6] by grafting mouse complementarity-determining regions (CDRs) with humanized framework sequences [7].

Example 3. CAR Lentivirus

2.5×10⁷ HEK293FT cells (Thermo Fisher) were seeded on 0.01% gelatin-coated 15 cm plates and cultured overnight in DMEM, 2% FBS, 1×pen/strep, and then transfected with the pPACKH1 Lentivector Packaging mix (System Biosciences, Palo Alto, Calif., USA) and 10 μg of the lentiviral vector using the NanoFect transfection reagent NF100 (Alstem, Richmond, Calif., USA). The next day the medium was replaced with fresh medium, and 48 h later, the lentivirus-containing medium was collected. The medium was cleared of cell debris by centrifugation at 2100×g for 30 min. The virus particles were collected by centrifugation at 112,000×g for 60 min at 4° C. using a SW28.1 rotor, suspended in serum-free DMEM medium, aliquoted, and frozen at −80° C.

Example 4. Peripheral Blood Mononuclear Cell (PBMC) Isolation from Whole Blood

Whole blood (Stanford Hospital Blood Center, Stanford, Calif.) was collected from individual or mixed donors (depending on the amount of blood required) in 10 mL Heparin vacutainers (Becton Dickinson). Approximately 10 ml of whole anti-coagulated blood was mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20 ml in a 50 ml conical centrifuge tube (PBS, pH 7/4, is without Ca2+/Mg2+). The layer of cells containing peripheral blood mononuclear cells (PBMC), seen at the diluted plasma/Ficoll interface was removed very carefully, avoiding any Ficoll, washed twice with PBS, and centrifuged at 200×g for 10 min at room temperature. Cells were counted with a hemocytomter. The PBMC were washed once with CAR-T media (AIM V-AlbuMAX(BSA)(Life Technologies), with 5% AB serum and 1.25 ug/mL amphotericin B (Gemini Bioproducts, Woodland, Calif.), 100 U/mL penicillin, and 100 ug/mL streptomycin) and used for experiments or were frozen at −80° C.

Example 5. T-Cell Activation from PBMC

The isolated cells (washed with 1×PBS (pH7.4), no Ca²⁺/Mg²⁺) were washed once in CAR-T media (AIM V-AlbuMAX(BSA)(Life Technologies), with 5% AB serum and 1.25 μg/mL amphotericin B (Gemini Bioproducts, Woodland, Calif.), 100 U/mL penicillin, and 100 μg/mL streptomycin), in the absence of human interleukin-2 (huIL-2)(Invitrogen), at a concentration of 5×10⁵ cells/mL. The cells were resuspended to a final concentration of 5×10⁵ cells/mL in CAR-T medium with 300 U/mL huIL2. The PBMC were activated at a 1:1 CD3-CD28 bead-to-cell ratio.

Example 6. T-Cell Transduction and Expansion

Following activation of PBMC, cells were incubated for 24 hr at 37° C., 5% CO₂. To each well of 1×10⁶ cells add 5×10⁶ lentivirus, and 2 μL/mL of media of Transplus (Alstem, Richmond, Calif.) (a final dilution of 1:500). Cells were incubated for an additional 24 hours before repeating addition of virus. Cells were then grown in the continued presence of 300 U/ml of IL-2 Fresh medium with IL-2 for a period of 12-14 days (total incubation time is dependent on the final umber of CAR-T cells required). Cell concentrations were analyzed every 2-3 days, with media being added at that time to dilute the cell suspension to 1×10⁶ cells/ml.

Example 7. Flow Cytometry (FACS)

To measure CAR expression, 0.25 million cells were suspended in 100 μL of buffer (PBS (phosphate buffered saline) containing 2 mM EDTA pH 8 and 0.5% BSA) and incubated on ice with 1 μL of human serum for 10 min. Diluted primary antibody biotin-conjugated goat anti-mouse (Fab)₂ or anti-human (Fab)₂ was used with cells for 30 min at 4° C., and after washing, the secondary antibody was added with APC-conjugated mouse α-human CD3 antibody and PE-conjugated streptavidin at 1:100 dilution for 30 min incubation at 4° C. The cells were rinsed with 3 mL of washing buffer, then stained for 10 min with 7-AAD, suspended in the buffer, and acquired on a FACSCalibur (BD Biosciences, San Jose, Calif., USA). Cells were analyzed first for light scatter versus 7-AAD staining, then the 7-AAD-live gated cells were plotted for anti-CD3 staining versus CAR+ staining with anti-(Fab)₂ antibodies.

Example 8. Cytotoxicity (RTCA)

Adherent target cells (CHO-CD37; CHO; Hela-CD37 or Hela) were seeded into 96-well E-plates (Acea Biosciences, San Diego, Calif., USA) at 1×104 cells per well and monitored in culture overnight with the impedance-based real-time cell analysis (RTCA) xCELLigence system (Acea Biosciences). The next day, the medium was removed and re-placed with AIM V-AlbuMAX medium containing 10% FBS±1×105 effector cells (CAR-T cells or non-transduced T cells) in triplicate. The cells in the E-plates were monitored for another 24-48 h with the RTCA system, and impedance was plotted over time. Cytotoxicity was calculated as (impedance of target cells without effector cells-impedance of target cells with effector cells)×100/impedance of target cells without effector cells.

Example 9. ELISA for Detection IFN-Gamma

Nonadherent target cells (Raji, MMTS, K562) were cultured with the effector cells (CAR-T cells or non-transduced T cells) at a 1:1 ratio (1×104 cells each) in U-bottom 96-well plates with 200 μL of AIM V-AlbuMAX medium containing 10% FBS, in triplicate. After 16 h, the top 150 μL of the medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells. The top 120 μL of supernatant was transferred to a new 96-well plate and analyzed by ELISA for human IFN-γ levels using a kit from R&D Systems (Minneapolis, Minn., USA) according to the manufacturer's protocol. The supernatant after RTCA with adherent target cells was collected and analyzed as above.

Example 10. Mouse Tumor Xenograft Model and Imaging

Six-week-old male NSG mice (Jackson Laboratories, Bar Harbor, Me., USA) were housed in accordance with the Institutional Animal Care and Use Committee (IACUC) (#LUM-001). Each mouse was injected subcutaneously on day 0 with 100 μL of 5×10⁵ Raji-luciferase positive cells in sterile serum-free medium. The next day 1×10⁷ CAR-T cells in serum-free medium were injected intravenously. Imaging was done using Raji-luciferase positive cells after luciferin injection with Xenogen Ivis System. Quantification was done by measuring photons/sec signals. A Kaplan-Myer survival curve was done based on mice survival data.

Example 11. Statistical Analysis

Data were analyzed and plotted with Prism software (GraphPad V7, San Diego, Calif., USA). Comparisons between two groups were performed by unpaired Student's t-test; one or two-way ANOVA, followed by Sidak or Dunnett's tests for multiple comparisons. The p-value <0.05 was considered significant.

Sequences Example 12. Two Humanized CD37 scFv Sequences

Two sequences of VH and VL and ScFv of humanized CD37 (hCD37) antibody were selected for their high activities in CAR scFv format against CD37-positive cells.

The two sequences of ScFv of humanized CD37 (hCD37) antibody are shown below. The structure of hCD37 scFv is: VH-linker-VL. Linker is G4Sx3. The bold shows the nucleotide sequence; the underlined shows the nucleotide sequence of VL; in between (shown in italics font) is the nucleotide sequence encoding a linker.

(a) hCD37 ScFv (#1) Humanized CD37 ScFv #1, nucleotide sequence (VH bold, linker G4S, VL underlined): (SEQ ID NO: 1) GAA GTA CAA CTC GTC GAG TCC GGC GGG GGA CTG GTA CAG CCC GGA CGG TCC CTG AGA CTT AGT TGT ACG GCT TCT GGT TTC ACG TTT TCC GAC TAT TGG ATG AAT TGG GTG AGG CAA GCG CCC GGT AAA GGC CTG GAA TGG GTG GGA CAA ATT CGC GAT AAG CCG TAT AAT TAC GAA ACA TTC TAC AGC GAC TCT GTC AAG GGT AGA TTC ACA ATC TCC CGG GAT GAC AGT AAA TCC ATT GCA TAC CTC CAG ATG AAC TCT CTC AAA ACC GAG GAT ACA GCT GTA TAT TAT TGC ACT GGG AGT TTC GCC TAC TGG GGA GCT GGG ACG ACG GTA ACG GTA TCC TCA GGT GGC GGT GGA AGC GGT GGT GGA GGT AGT GGA GGG GGT GGG AGT GCC ATT AGG ATG ACT CAG AGC CCC AGT TCT TTC AGT GCA TCA ACA GGA GAC AGA GTA ACG ATA ACG TGC CGG GCA AGC GGT AAC ATC CAT AAT TAC CTT GCG TGG TAC CAA CAG AAA CCT GGT AAA GCG CCG AAA CTT CTC ATT TAC AAT GCT AAG ACC CTT CCC TCC GGA GTT CCC TCT AGG TTT AGT GGC TCA GGT AGC GGG ACC GAC TTT ACC TTG ACA ATC AGT TGC CTG CAA TCA GAA GAC TTT GCA ACT TAC TAC TGC CAA CAG TAT TGG AGC ACC CCC TAT ACG TTT GGC GGC GGT ACT AAA CTG GAA ATC CGC Humanized CD37 ScFv #1, amino acid sequence (SEQ ID NO: 2) EVQLVESGGGLVQPGRSLRLSCTASGFTFSDYWMNWVRQAPGKGLEWVGQIR DKPYNYETFYSDSVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTGSFAYWG AGTTVTVSSGGGGSGGGGSGGGGSAIRMTQSPSSFSASTGDRVTITCRASGNIHNYL AWYQQKPGKAPKLLIYNAKTLPSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQY WSTPYTFGGGTKLEIR Humanized CD37 VH #1, amino acid sequence, (SEQ ID NO: 3) EVQLVESGGGLVQPGRSLRLSCTASGFTFSDYWMNWVRQAPGKGLEWVGQIR DKPYNYETFYSDSVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTGSFAYWG AGTTVTVSS Humanized CD37 VL #1 amino acid sequence (SEQ ID NO: 4) AIRMTQSPSSFSASTGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLIYNAKTLPSGV PSRFSGSGSGTDFTLTISCLOSEDFATYYCQQYWSTPYTFGGGTKLEIR The linker amino sequence is 3xG4S (SEQ ID NO: 5): GGGGSGGGGSGGGGS (b) hCD37 ScFv (#2) Humanized CD37 ScFv #2, nucleotide sequence (VH bold, linker G4S in italics, VL underlined): (SEQ ID NO: 6) CAGGTGCAGCTTGTCGAGAGTGGAGGTGGCGTCGTTCAACCTGGTAGAAGT TTGCGGCTCTCATGTGCGGCCTCTGGATTTACTTTTTCTGACTACTGGATGA ACTGGGTCAGGCAAGCTCCAGGTAAGGGGCTTGAATGGGTGGCGCAGATCC GGGATAAGCCATACAACTACGAGACGTTCTACTCAGACAGTGTAAAGGGCA GATTTACTATAAGTAGAGATAATTCAAAGAACACGCTCTATCTTCAGATGAA TTCATTGAGAGCGGAGGACACGGCGGTGTACTACTGTACTGGCAGCTTTGC GTACTGGGGAGCTGGGACAACGGTAACGGTATCTTCT GGAGGAGGAGGTAGT GGCGGGGGTGGCTCTGGAGGTGGCGGCTCT GATATACAAATGACTCAAAGTCCAT CTAGTCTCAGCGCCAGCGTGGGCGACAGGGTCACTATAACGTGTCGAGCGTCTG GCAATATACACAATTACTTGGCGTGGTATCAGCAAAAACCGGGAAAAGCGCCGA AGCTGCTCATATATAATGCGAAAACTTTGCCATCAGGCGTTCCTTCCCGGTTCTC AGGCTCCGGTTCTGGAACGGACTTTACTCTCACTATCAGTAGCCTTCAGCCAGAG GACTTCGCCACTTACTATTGCCAGCAATATTGGTCTACTCCTTACACCTTTGGGGG AGGGACCAAATTGGAAATCCGC Humanized CD37 ScFv #2, amino acid sequence (SEQ ID NO: 7) QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYWMNWVRQAPGKGLEWVAQIRDKPYN YETFYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTGSFAYWGAGTTVTVSS G GGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLI YNAKTLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWSTPYTFGGGTKLEIR Humanized CD37 VH #2, amino acid sequence, (SEQ ID NO: 8) QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYWMNWVRQAPGKGLEWVAQIR DKPYNYETFYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTGSFAYWG AGTTVTVSS Humanized CD37 VL #2 amino acid sequence, (SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLIYNAKTLPSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWSTPYTFGGGTKLEIR

The linker amino sequence is the same 3xG4S (SEQ ID NO: 5)

Example 13A. Humanized CD37 CAR Sequence #1 (PMC762 CAR or PMC985CAR)

The full length humanized CD37 CAR (PMC762) is shown below: signaling peptide, humanized CD37scFv (VH-linker-VL), Hinge, transmembrane domain, 41BB and CD3 domains.

<CD8 alpha signaling peptide +Nhe I site:> Nucleotide sequence (SEQ ID NO: 10) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC CGCCAGGCCGgctagc Amino acid sequence (+NheI site at 3′ end for cloning: AS), (SEQ ID NO: 11) MALPVTALLLPLALLLHAARPAS <CD37 ScFv, #1> See Example 12a <Xho site> Nucleotide sequence Ctcgag Amino acid sequence LE <spacer> Nucleotide sequence AAGCCC Amino acid sequence KP <CD8 alpha hinge> Nucleotide sequence (SEQ ID NO: 12) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCAGTGAT Amino acid sequence (SEQ ID NO: 13) TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD <CD28 Transmembrane domain> Nucleotide sequence (SEQ ID NO: 14) Aagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacag tggcctttattattttctgggtg Amino acid sequence, (SEQ ID NO: 15) KPFWVLVVVGGVLACYSLLVTVAFIIFWV <41BB> Nucleotide sequence (SEQ ID NO: 16) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA TGTGAACTG Amino acid sequence (SEQ ID NO: 17) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL <CD3> Nucleotide sequence (SEQ ID NO: 18) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAA CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCA GGCCCTGCCCCCTCGCTAA Amino acid sequence (SEQ ID NO: 19) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR < Humanized CD37-CAR, PMC762> Nucleotide sequence (SEQ ID NO: 20) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCGgctagc GAA GTA CAA CTC GTC GAG TCC GGC GGG GGA CTG GTA CAG CCC GGA CGG TCC CTG AGA CTT AGT TGT ACG GCT TCT GGT TTC ACG TTT TCC GAC TAT TGG ATG AAT TGG GTG AGG CAA GCG CCC GGT AAA GGC CTG GAA TGG GTG GGA CAA ATT CGC GAT AAG CCG TAT AAT TAC GAA ACA TTC TAC AGC GAC TCT GTC AAG GGT AGA TTC ACA ATC TCC CGG GAT GAC AGT AAA TCC ATT GCA TAC CTC CAG ATG AAC TCT CTC AAA ACC GAG GAT ACA GCT GTA TAT TAT TGC ACT GGG AGT TTC GCC TAC TGG GGA GCT GGG ACG ACG GTA ACG GTA TCC TCA GGT GGC GGT GGA AGC GGT GGT GGA GGT AGT GGA GGG GGT GGG AGT GCC ATT AGG ATG ACT CAG AGC CCC AGT TCT TTC AGT GCA TCA ACA GGA GAC AGA GTA ACG ATA ACG TGC CGG GCA AGC GGT AAC ATC CAT AAT TAC CTT GCG TGG TAC CAA CAG AAA CCT GGT AAA GCG CCG AAA CTT CTC ATT TAC AAT GCT AAG ACC CTT CCC TCC GGA GTT CCC TCT AGG TTT AGT GGC TCA GGT AGC GGG ACC GAC TTT ACC TTG ACA ATC AGT TGC CTG CAA TCA GAA GAC TTT GCA ACT TAC TAC TGC CAA CAG TAT TGG AGC ACC CCC TAT ACG TTT GGC GGC GGT ACT AAA CTG GAA ATC CGC ctcgagAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCAT CGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGG CGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagcccttttgggtgctggtggtggttgg tggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgAAACGGGGCA GAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCA GCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG AGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGG ATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTC AGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GCTAA Amino acid sequence of humanized CD37 CAR (SEQ ID NO: 21) MALPVTALLLPLALLLHAARPASEVQLVESGGGLVQPGRSLRLSCTASGFTFSDY WMNWVRQAPGKGLEWVGQIRDKPYNYETFYSDSVKGRFTISRDDSKSIAYLQM NSLKTEDTAVYYCTGSFAYWGAGTTVTVSS GGGGSGGGGSGGGGS AIRMTQSPSS FSASTGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLIYNAKTLPSGVPSRFSGSGSG TDFTLTISCLQSEDFATYYCQQYWSTPYTFGGGTKLEIRLEKPTTTPAPRPPTPAPTIAS QPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWV KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 13B. Humanized CD37 CAR Sequence #2 (PMC910 CAR)

The structure and the sequences of this humanized CD37 CAR sequence #2 (PMC910) are similar to those of Example 13A, except the scFv is hCD37 #2 (see Example 12 (b))

The nucleotide sequence of PMC910 CAR is shown below (SEQ ID NO: 22) atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggctagccaggtgcagcttgtcgaga gtggaggtggcgtcgttcaacctggtagaagtttgcggctctcatgtgcggcctctggatttactttttctgactactggatgaactgggtc aggcaagctccaggtaaggggcttgaatgggtggcgcagatccgggataagccatacaactacgagacgttctactcagacagtgta aagggcagatttactataagtagagataattcaaagaacacgctctatcttcagatgaattcattgagagcggaggacacggcggtgta ctactgtactggcagctttgcgtactggggagctgggacaacggtaacggtatcttctggaggaggaggtagtggcgggggtggctct ggaggtggcggctctgatatacaaatgactcaaagtccatctagtctcagcgccagcgtgggcgacagggtcactataacgtgtcgag cgtctggcaatatacacaattacttggcgtggtatcagcaaaaaccgggaaaagcgccgaagctgctcatatataatgcgaaaactttg ccatcaggcgttccttcccggttctcaggctccggttctggaacggactttactctcactatcagtagccttcagccagaggacttcgcca cttactattgccagcaatattggtctactccttacacctttgggggagggaccaaattggaaatccgcctcgagaagcccaccacgacg ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgagccggccagcggc ggggggcgcagtgcacacgagggggctggacttcgccagtgataagcccttttgggtgctggtggtggttggtggagtcctggcttg ctatagcttgctagtaacagtggcctttattattttctgggtgaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgag accagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagtt cagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagta cgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctcta caatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacg atggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa The amino acid sequence of PMC910 CAR is shown below (SEQ ID NO: 23) MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYW MNWVRQAPGKGLEWVAQIRDKPYNYETFYSDSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCTGSFAYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLIYNAKTLPSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQYWSTPYTFGGGTKLEIRLEKPTTTPAPRPPTPAPTIASQPL SLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 14A. Bispecific CD19-hCD37 CAR Sequence (PMC 930)

The scheme of bi-specific CD19-humanized CD37-CAR construct is shown on FIG. 2 . The scFV is hCD37 antibody #1. Lentiviral vector was used for cloning of CD19-humanized CD37 scFv CAR sequences.

The following nucleotide and amino acid sequences show GM-CSF receptor alpha signaling peptide, CD19 VL-Linker (G4S)-humanized CD37 ScFv (hCD37 VH-Linker-hCD37 VL); G4S linker; CD19 VH; CD8 hinge; CD8 transmembrane; 41BB costimulatory domain; CD3 zeta activation domains (FIG. 2B).

<GM-CSF receptor alpha leader> Nucleotide sequence (SEQ ID NO: 24) Atgctgctgctcgtgacaagcctgctgctgtgcgagctgccccaccctgcctttctgctgatcccc Amino acid sequence (SEQ ID NO: 25) MLLLVTSLLLCELPHPAFLLIP <CD19 VL> Nucleotide sequence (SEQ ID NO: 26) gacatccagatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccatcagctgcagagccagccaggac atcagcaagtacctgaactggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccacaccagcagactgcacagcgg cgtgcccagcagattttctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgctacgtac ttttgtcagcagggaaacacgcttccatacaccttcggcggcggtacgaagttggagatcacg Amino acid (SEQ ID NO: 27) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT <G4S linker> (SEQ ID NO: 5) <hCD37 ScFv, #1>=hCD37 VH-linker-hCD37 VL (Same as Example 12A) <Linker G4S> (SEQ ID NO: 5) <CD19 VH> Nucleotide Sequence (SEQ ID NO: 28)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Amino acid sequence (SEQ ID NO: 29) EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS <two amino-acids spacer> Nucleotide sequence AGC GGG Amino acid sequence SG <CD8 hinge> Nucleotide sequence (SEQ ID NO: 30) accacgacgcccgcccctagacccccgacgcccgctccgactatagegagccaacctctcagcctgaggcctgaagcatgtcgacc agcagcaggaggggcagtacacaccaggggcctggattttgcctgtgat Amino acid sequence (SEQ ID NO: 31) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD <CD8 transmembrane domain> Nucleotide sequence (SEQ ID NO: 32) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc Amino acid sequence (SEQ ID NO: 33) IYIWAPLAGTCGVLLLSLVITLYC

<41BB Costimulatory Domain>

See Example 13A

<CD3 Zeta>

See Example 13A

The full length CD19-humanized-CD37 CAR (PMC930) is shown below: GM-CSF receptor alpha signaling peptide underlined; CD19 VL in bold; G4S linker italics underlined; humanized CD37 ScFv (VH-linker-VL); linker G4S underlined italics; CD19 VH bold, italics; SG amino-acids; then CD8 alpha hinge, in bold, underlined; CD8 alpha 15 transmembrane domain regular font, underlined; then 4-1BB domain in bold; CD3 activation domain, regular font.

The nucleotide sequence of bispecific CD19-humanized CD37-CAR (PMC930 CAR) is shown below (SEQ ID NO: 34).

atgctgctgctcgtgacaagcctgctgctgtgcgagctgccccaccctgcctttctgctgat cccc gacatccagatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccat cagctgcagagccagccaggacatcagcaagtacctgaactggtatcagcagaaacccgacg gcaccgtgaagctgctgatctaccacaccagcagactgcacagcggcgtgcccagcagattt tctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatat cgctacgtacttttgtcagcagggaaacacgcttccatacaccttcggcggcggtacgaagt tggagatcacg GGC GGA GGA GGG AGT  GAA GTA CAA CTC GTC GAG TCC GGC GGG GGA CTG GTA CAG CCC GGA CGG TCC CTG AGA CTT AGT TGT ACG GCT TCT GGT TTC ACG TTT TCC GAC TAT TGG ATG AAT TGG GTG AGG CAA GCG CCC GGT AAA GGC CTG GAA TGG GTG GGA CAA ATT CGC GAT AAG CCG TAT AAT TAC GAA ACA TTC TAC AGC GAC TCT GTC AAG GGT AGA TTC ACA ATC TCC CGG GAT GAC AGT AAA TCC ATT GCA TAC CTC CAG ATG AAC TCT CTC AAA ACC GAG GAT ACA GCT GTA TAT TAT TGC ACT GGG AGT TTC GCC TAC TGG GGA GCT GGG ACG ACG GTA ACG GTA TCC TCA GGT GGC GGT GGA AGC GGT GGT GGA GGT AGT GGA GGG GGT GGG AGT GCC ATT AGG ATG ACT CAG AGC CCC AGT TCT TTC AGT GCA TCA ACA GGA GAC AGA GTA ACG ATA ACG TGC CGG GCA AGC GGT AAC ATC CAT AAT TAC CTT GCG TGG TAC CAA CAG AAA CCT GGT AAA GCG CCG AAA CTT CTC ATT TAC AAT GCT AAG ACC CTT CCC TCC GGA GTT CCC TCT AGG TTT AGT GGC TCA GGT AGC GGG ACC GAC TTT ACC TTG ACA ATC AGT TGC CTG CAA TCA GAA GAC TTT GCA ACT TAC TAC TGC CAA CAG TAT TGG AGC ACC CCC TAT ACG TTT GGC GGC GGT ACT AAA CTG GAA ATC CGC GGT GGT GGA GGG AGC  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 AGC GGG  ACC ACG ACG CCC GCC CCT AGA CCC CCG ACG CCC GCT CCG ACT ATA GCG AGC CAA CCT CTC AGC CTG AGG CCT GAA GCA TGT CGA CCA GCA GCA GGA GGG GCA GTA CAC ACC AGG GGC CTGGAT TTT GCC TGT GATatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttat caccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga gaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaa ggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcaggg ccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggaca agagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggc ctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaagg cgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaagg acacctacgacgcccttcacatgcaggccctgccccctcgctaa The amino acid sequence of bispecific CD19-humanized CD37-CAR (PMC930) is shown below. (SEQ ID NO: 35) MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSEVQLVESGGGLVQPGRSLRLSCTASGFTFSDYWMNWVRQA PGKGLEWVGQIRDKPYNYETFYSDSVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYY CTGSFAYWGAGTTVTVSSGGGGSGGGGSGGGGSAIRMTQSPSSFSASTGDRVTITCR ASGNIHNYLAWYQQKPGKAPKLLIYNAKTLPSGVPSRFSGSGSGTDFTLTISCLQSED FATYYCQQYWSTPYTFGGGTKLEIRGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGV SLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNS LQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSSGTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 14B. Bispecific CD19-hCD37 CAR Sequence (PMC 1046)

The structure and the sequences of this bispecific CD19-hCD37 CAR sequence #2 (PMC1046) are similar to those of Example 13A, except the scFv of hCD37 (#2) is used in the CAR.

Nucleotide sequence of PMC1046 (SEQ ID NO: 36) atgctgctgctcgtgacaagcctgctgctgtgcgagctgccccaccctgcctttctgctgatccccgacatcca gatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccatcagctgcagagccagccaggacatcagcaa gtacctgaactggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccacaccagcagactgcacagcggcgtgccca gcagattttctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatc GCT ACG TAC TTT TGT CAG CAG GGA AAC ACG CTT CCA TAC ACC TTC GGC GGC GGT ACG AAG TTG GAG ATCACG GGC GGA GGA GGG AGT CAGGTGCAGCTTGTCGAGAGTGGAGGTGGCGTCGTTCAACCTGGTAGAAGTTTGC GGCTCTCATGTGCGGCCTCTGGATTTACTTTTTCTGACTACTGGATGAACTGGGTC AGGCAAGCTCCAGGTAAGGGGCTTGAATGGGTGGCGCAGATCCGGGATAAGCCA TACAACTACGAGACGTTCTACTCAGACAGTGTAAAGGGCAGATTTACTATAAGTA GAGATAATTCAAAGAACACGCTCTATCTTCAGATGAATTCATTGAGAGCGGAGG ACACGGCGGTGTACTACTGTACTGGCAGCTTTGCGTACTGGGGAGCTGGGACAA CGGTAACGGTATCTTCTGGAGGAGGAGGTAGTGGCGGGGGTGGCTCTGGAGGTG GCGGCTCTGATATACAAATGACTCAAAGTCCATCTAGTCTCAGCGCCAGCGTGGG CGACAGGGTCACTATAACGTGTCGAGCGTCTGGCAATATACACAATTACTTGGCG TGGTATCAGCAAAAACCGGGAAAAGCGCCGAAGCTGCTCATATATAATGCGAAA ACTTTGCCATCAGGCGTTCCTTCCCGGTTCTCAGGCTCCGGTTCTGGAACGGACTT TACTCTCACTATCAGTAGCCTTCAGCCAGAGGACTTCGCCACTTACTATTGCCAG CAATATTGGTCTACTCCTTACACCTTTGGGGGAGGGACCAAATTGGAAATCCGC GGT GGT GGA GGG AGC GAGGTC AAA CTG CAG GAA TCC GGA CCA GGG CTT GTA GCC CCC TCA CAA AGC CTC AGC GTG ACT TGT ACA GTGAGC GGC GTT AGT CTG CCG GAC TAT GGA GTT TCT TGG ATT CGG CAA CCG CCC AGA AAA GGG CTG GAA TGGCTT GGT GTT ATA TGG GGC TCA GAA ACT ACC TAT TAC AAC AGT GCT CTC AAA AGC CGG CTC ACA ATA ATAAAG GAT AAT AGC AAA TCT CAA GTT TTC CTG AAA ATG AAC TCT CTG CAA ACA GAT GAC ACG GCG ATC TACTAC TGC GCG AAA CAC TAC TAT TAC GGC GGC AGC TAC GCC ATG GAC TAT TGG GGG CAG GGG ACG TCA GTGACA GTG TCT AGC AGC GGG ACC ACG ACG CCC GCC CCT AGA CCC CCG ACG CCC GCT CCG ACT ATA GCG AGCCAA CCT CTC AGC CTG AGG CCT GAA GCA TGT CGA CCA GCA GCA GGA GGG GCA GTA CAC ACC AGG GGC CTGGAT TTT GCC TGTgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgt cactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactac tcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgc agacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggac aagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaa agataagatggoggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccaggg tctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa Amino acid sequence of PMC1046 (SEQ ID NO: 37) MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYWMNWVRQ APGKGLEWVAQIRDKPYNYETFYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCTGSFAYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT CRASGNIHNYLAWYQQKPGKAPKLLIYNAKTLPSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYWSTPYTFGGGTKLEIRGGGGSEVKLQESGPGLVAPSQSLSVTCTVS GVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKM NSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSSGTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Results Example 15. Humanized CD37-CAR-T Cells (PMC762) Specifically Targeted CD37-Positive Cells

We humanized CD37 VH and VL, as described in Materials and Methods, and generated lentiviral humanized CD37 CAR with a 4-1BB costimulatory domain and CD3 activation domain, called hCD37 CAR (PMC762). Surface expression of the CAR was detected by FACS with both anti-mouse Fab (72% positive) and anti-Human Fab (92% positive). In real-time cytotoxicity assay against CHO-CD37 and CHO cells, humanized anti-CD37 CAR-T cells (PMC762) effectively killed CHO-CD37 cells and demonstrated limited or no killing of CHO cells (FIG. 3A). Cytotoxicity of humanized CD37 CAR-T cells against CHO-CD37 (95.3%±0.8%) was significantly higher than non-transduced T cells (17.5%±1.3%) or mock CAR-T cells (FIG. 3B). The hCD37-CAR-T cells secreted significantly higher levels of IFN-gamma with CD37-positive target cells than with CD37-negative cells (FIG. 3C). Thus, humanized CD37-CAR-T cells specifically target CD37-positive cells.

Example 16. Bispecific Humanized CD37-CD19 CAR-T Cells (PMC930) Specifically Targeted CD37-Positive Cells

We tested the efficacy of bi-specific humanized hCD37-CD19 CAR-T cells (PMC930) in vitro. To generate bi-specific humanized CD37-CD19 CAR-T cells, we used the following design as shown in FIG. 2B with humanized CD37 ScFv. These CAR-T cells had a surface expression of CAR as detected by FACS with anti-human Fab antibodies (not shown). Real-time cytotoxicity assays were performed against CHO-CD37 and CHO cells (FIG. 4A) and against Hela-CD19 and Hela cells (FIG. 4B). Killing by bispecific hCD37-CD19 CAR-T cells was compared to CAR-T cells expressing monospecific hCD37 CAR or CD19 CAR. Bi-specific hCD37-CD19 CAR-T cells killed CHO-CD37 as effective as single hCD37-CAR-T cells and did not kill CHO cells (FIG. 4A).

The hCD37-CD19 CAR-T cells also killed Hela-CD19 target cells and did not kill Hela cells (FIG. 4B). As expected, single hCD37-CAR-T cells did not kill Hela-CD19 cells. The hCD37-CD19 CAR-T cells and hCD37-CAR-T cells secreted significantly higher levels of IFN-gamma against CHO-CD37 cells versus CHO cells (FIG. 4C). Both hCD37-CD19 and CD19-CAR-T cells secreted significantly higher levels of IFN-gamma against Hela-CD19 target cells but not against Hela cells (FIG. 4D).

In separate co-culture experiments, IFN-γ release against Raji cells or MM1S cells was measured (FIG. 4E). Both CD37-CD19 CAR-T cells and CD19 CAR-T cells had significantly more IFN-γ release than humanized CD37 CAR-T cells, mock CAR-T cells, and non-transduced T cells (p<0.0001, Tukey's test) (FIG. 4E). The secretion of IFN-gamma was significantly higher for CD37-CD19-CAR-T cells against Raji cells than against MM1S cells.

Thus, hCD37-CD19 CAR-T cells demonstrate high and specific efficacy against CD37 and CD19-positive target cells in vitro.

Example 17. Comparison PMC985 (762), PMC910 and PMC930 and PMC1046 CAR-T Cells In Vitro

We compared humanized CD37 CAR (PMC985) and bispecific CD19-CD37 CAR (PMC930) with another humanized CD37 (PMC910) and bispecific CD19-CD37 CAR (PMC1046) by RTCA. PMC985 CAR and PMC762 CAR have the same scFV and the same sequences; the only difference between PMC 985 CAR and PMC762 CAR is that the vector for creating PMC762 has AmpR, whereas the vector for creating PMC985 has Kan-R. We also used PMC1009 CD19-41BB-CD3 CAR-T cells. We prepared freshly viruses and used equal number of CAR+ cells and performed RTCA assay using CHO-CD37, CHO and Hela-CD19, Hela cells. PMC930 CAR-T cells were slightly more cytotoxic than PMC1046 in RTCA assay against Hela-CD19 cells (FIG. 5 ).

Example 18. Bispecific Humanized CD37-CD19 CAR-T Cells (PMC930) Inhibited Raji

Lymphoma Xenograft Tumor Growth and Prolong Mice Survival

At first, we tested the efficacy of CD37-CAR-T cells (PMC762) in vivo and performed survival analysis using a Raji-xenograft tumor model after an injection of humanized CD37-CAR-T cells (FIG. 6 ). Humanized CD37-CAR-T cells prolonged mouse survival as well as CD19-CAR-T cells (FIG. 6 ).

To test the efficacy of the bispecific humanized CD37-CD19 CAR-T cells in vivo, Nod Scid Gamma, NSG mice were injected with 5×10⁵ Raji-Luc cells followed 24 h later with 1×10⁷ humanized CD37-CD19 CAR-T cells, mock CAR-T cells, or vehicle. Tumor luminescence was detected in mice treated with mock CAR-T cells or vehicle but not in mice treated with CD37-CD19 CAR-T cells (FIG. 7A). Tumor luminescence in CD37-CD19 CAR-T cell treated mice was significantly lower than in mock CAR-T cell treated mice (FIG. 7B). Survival of CD37-CD19 CAR-T cell treated group was significantly longer (>75 days) (log-rank test p<0.0001) than vehicle (18 days) and mock CAR-T cell treated groups (FIG. 7C). Thus, humanized CD37 CAR-T cells and bi-specific hCD37-CD19 CAR-T cells are efficacious in the model in vivo.

REFERENCES

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1. A humanized anti-human CD37 antibody or an antigen-binding fragment thereof comprising (i) VH having the amino acid sequence of SEQ ID NO: 3 and VL having the amino acid sequence of SEQ ID NO:
 4. 2. The humanized anti-human CD37 antibody or an antigen-binding fragment thereof, which is scFv.
 3. The humanized scFv of claim 2, which has the amino acid sequence of SEQ ID NO:
 2. 4-18. (canceled) 